How Does ELISA Testing Work? A Versatile Immunoassay

ELISA (enzyme-linked immunosorbent assay) is an indispensable laboratory test used to detect antibodies, antigens, proteins, and other substances for a wide range of diagnostics and research applications.

How Does ELISA Testing Work?

ELISA leverages immunoassay principles to identify targeted analytes:

• Coating the Surface with Capture Antibodies: The ELISA plate surface is coated with antibodies specific to the substance being measured. This “captures” matching analytes.
• Blocking Sites: Blocking proteins attach to any open spaces on the plate to prevent non-specific binding later.
• Adding Fluid Samples: Patient serum, urine, saliva or other fluids containing the analyte are added, so they can bind to the capture antibodies.
• Washing: Washing removes unbound components, leaving only captured analytes on the plate.
• Adding Detecting Antibodies: Antibodies coupled with enzymes are added that attach to the bound analytes. Common enzymes used are horseradish peroxidase (HRP) or alkaline phosphatase (AP).
• Substrate Solution: When the substrate is added, reactions with the coupled enzymes produce measurable colour changes or fluorescence proportional to the concentration of analyte present.
• Stopping Reaction: A stopping solution halts the reaction to allow quantification based on the colour intensity or fluorescence.

ELISA Test Types

There are several ELISA variants designed to test different samples for specific analytes:

1. Direct ELISA (Detects Antigen): Capture antibody binds antigen in the sample. Detecting antibodies also binds antigens. Enzyme reaction signals presence.
2. Indirect ELISA (Detects Antibody): Antigen coated to surface. If antibodies are present in the sample, they bind antigen. An added secondary antibody with enzyme binds the sample antibody, signalling presence.
3. Sandwich ELISA (Detects Antigen): The surface has a capture antibody. Sample antigen binds capture antibody. Detecting antibodies also binds antigens, allowing enzymes to act on the substrate.
4. Competitive ELISA (Small Antigens/Haptens): Both sample antigens and labelled antigens compete for binding sites on antibodies coated on a plate. More antigens in the sample = less labelled antigen binding and weaker signal.

Advantages of ELISA Testing

• Highly specific and sensitive for detection within a sample. Can quantify analyte concentration.
• Amenable to high throughput automated analysis of multiple samples.
• Relatively inexpensive compared to other antibody detection methods.
• Allows standardization of results across labs when developed properly.
• Versatile: can detect and analyse many biomolecules, pathogens, drugs, allergens etc.
• Samples are stable for days, allowing batch analysis, unlike other rapid immunodiagnostics.

Analytes Detected by ELISA

ELISA allows detection of many antigens or antibodies, including:

• Viruses: HIV, hepatitis, herpesvirus, influenza
• Bacteria and parasites: Lyme, syphilis, toxoplasmosis
• Hormones: insulin, growth hormone, thyroid hormones
• Cancer biomarkers: PSA, CA-125, AFP
• Serum antibodies: rheumatoid factor, ANA, allergens
• Therapeutic drugs: monitoring levels and efficacy
• Biomolecules: cytokines, adhesion molecules, receptors

Applications of ELISA Testing

Key uses of ELISA assays include:

• Infectious disease diagnosis: HIV, Lyme, Chagas
• Allergy testing: identify IgE antibodies to allergens
• Oncology screening: tumour markers like PSA or CA-125
• Endocrinology: fertility, growth disorders, adrenal function
• Autoimmunity testing: celiac disease, lupus, rheumatoid arthritis
• Toxicology: therapeutic drug monitoring, drug abuse screening
• Biotechnology: recombinant protein analysis, epitope mapping
• Food safety: detecting spoiled, contaminated, or allergic food

FAQs About ELISA Testing

ELISA or enzyme-linked immunosorbent assay leverages antibodies coupled to colour reactions to detect biomolecules.

What does ELISA detect?

ELISA detects the presence of a specific analyte like an antigen, antibody, or protein by using antibodies coupled to enzymatic colour reactions.

What types of samples can be used for ELISA?

ELISA can be performed on serum, plasma, urine, saliva, cell culture supernatant and other biological fluid samples containing the analyte.

What diseases are diagnosed using ELISA?

ELISA assays diagnose infectious diseases like HIV and Lyme, autoimmunity disorders like celiac disease, cancers through tumour markers, and allergies.

Why is ELISA useful?

ELISA offers high sensitivity and specificity along with ease of automation, making it a versatile platform for detecting and quantifying numerous biomolecules.

What equipment is used in ELISA?

Key tools are microwell plates to bind reagents, precision pipettes, incubators, washers, photometers to measure reactions, and software to analyse data.

How ELISA Testing is Performed Step-By-Step?

ELISA testing involves the following key steps:

1. Coat wells with capture antibody specific to the target analyte.
2. Block open spaces in wells to prevent non-specific binding.
3. Add diluted patient samples and standards of known analyte concentration.
4. Wash wells to remove unbound components.
5. Add a detecting antibody-enzyme conjugate that binds to the captured analyte.
6. Wash again to remove the unbound conjugate.
7. Add a substrate solution that reacts with an enzyme to produce colour.
8. Add a stopping solution after the colour develops.
9. Measure absorbance or fluorescence signals.
10. Quantify analyte concentration by comparing signals to the standard curve.

With trained laboratory personnel, standardized reagents, and calibrated equipment, ELISA offers an accurate, sensitive lab test for in vitro detection of biomolecules.

Key Points About ELISA Immunoassay Testing

• Uses antibodies to capture and detect targeted antigen or antibody analytes
• Versatile technique to quantify numerous analytes from biological samples
• Direct, indirect, sandwich and competitive are the main ELISA variants
• Highly specific and sensitive to infectious diseases, cancers, allergies, hormones, drugs
• Allows automation of high-volume testing with rapid turnaround
• More affordable and quantitative than other antibody assays
• Standardization is critical for accurate, reproducible results

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